1. Field of the Invention
The present invention relates to mass spectrometry. In particular, the present invention relates to ion detection systems for mass spectrometry of biomolecules and other high molecular weight substances. Additionally, the present invention relates to improved ion detection systems for low molecular weight substances.
2. Background of Related Art
Accurate mass analysis of substances covering a wide range of molecular mass values is of increasing importance. In particular, accurate determination of molecular weights of proteins, and other biomolecules, is of paramount importance in biochemistry and modern biology. The molecular weight of a protein indicates its size, the possible presence of subunits (polymeric and monomeric molecular weights), and gives a rough idea of the number of component amino acids. An accurate method of high mass molecular weight determinations for proteins would be of special importance to the biotechnology field, since even rare proteins are now available by recombinant DNA techniques, and the first criteria of identity from batch to batch is the molecular weight of the protein.
In general, proteins range in molecular weight from 10,000 to 500,000 amu, but this range can be extended to include peptides (below 10,000 amu), or certain multimeric proteins (over 500,000 amu). At present, however, no accurate and efficient means is available for determination of biomolecular mass for the higher portion of the protein mass range, and in particular, for masses from 10,000 to 500,000 amu.
Determination of protein molecular weights by current methodologies, such as sedimentation, molecular sieving, gel electrophoresis, etc., present various special problems. The method of choice for determining protein molecular weights (weight average) has been sedimentation techniques in the ultracentrifuge. However, these techniques are technically cumbersome, slow and require the determination of other physical properties such as the partial specific volume of the protein. The accuracy of these methods can sometimes be as precise as to 10,000 amu, but more often are subject to much greater errors.
Mass spectrometry is one potential method for providing accurate determination of molecular weight of biomolecules and other molecules spanning a broad mass range. Mass spectrometry employs three functional aspects: sample ionization, mass analysis and ion detection. Progress has been achieved in all three major areas of mass spectral analysis. As a result reasonably effective measurements of certain biomolecules of mass below 10,000 amu have been achieved. Mass measurements for proteins as large as 25,000 amu have also been made using plasma desorption mass spectrometry. Nevertheless, the majority of protein structures have molecular weights from 10,000 to 200,000 amu and the need thus exists for new and improved methods in mass spectrometry to extend the range of mass analysis.
Presently available ion detection systems are not capable of efficient detection of ions in the mass range of from 10,000 to 500,000 amu, and in particular in the range of from 25,000 to 500,000 amu. Conventional means for detecting ions employed in mass spectrometry employ the impact of the ions at high velocity on a surface with the subsequent ejection of secondary electrons. These secondary electrons are detected by an electron multiplier resulting in an amplified signal pulse. Perhaps the most widely adopted method for the detection of low mass ions in mass spectrometry is the Channeltron Electron Multiplier (CEM), illustrated schematically in FIG. 1. This uses the direct impingement of incident ions on the surface of the detector to produce secondary electrons. Problems for the detection of high molecular weight ions derive from the well-known measured characteristics of these devices; in particular the reduction in the gain of CEM's with increasing M/Z of the bombarding ion. Now widely accepted, the phenomenon is attributed to the low yield of secondary electrons ejected by slow-moving, high mass molecules. R. J. Beuhler and L. Friedman, Threshold Studies of Secondary Electron Emission induced by Macro-Ion Impact on Solid Surfaces, Nucl. Instrum. Meth., 170, 309 (1980). Below a certain threshold velocity, detection may not be possible at all.
In an attempt to avoid the limitations on the primary ion source accelerating voltage, post-acceleration of the ions was introduced to increase the velocity of high mass ions. One approach to providing post-acceleration employs application of high voltages across the electron multipliers to accelerate the ions above the threshold. This is impractical, however, for voltages in excess of 3 to 4 kV due to intolerably low signal-to-noise levels. The disadvantages of such systems also include size, cost and complexity associated with bringing detector signals at high voltage to ground potential.
Another approach to post-acceleration ion detection for mass spectrometry is illustrated in FIG. 2. Post-acceleration of incident ion beams is provided by an intermediate conversion electrode (dynode) which can operate at high voltages. This circumvents one of the major problems associated with floating detectors at high voltages; for example, coupling the detector output signal to ground level electronics. Instead of directly bombarding the detector surface, the primary ions impact the dynode surface with an energy given by the voltage (V): EQU V=V.sub.a +V.sub.d
where V.sub.a is the ion source accelerating voltage and V.sub.d is the voltage applied to the dynode. Secondary electrons and negative ions ejected from the dynode surface are subsequently detected by conventional multipliers. Detection of high mass ions (50,000-100,000 amu) by post-acceleration methods will require dynode voltages of the same magnitude.
Various post-acceleration detector configurations have been reported and are commercially available from some manufacturers of magnetic instruments. One such detector is manufactured by JEOL Ltd. and is described in Evaluation of Post Acceleration Type High Sensitive Ion Detector For Mass Spectrometer, JEOL New, 21A (No. 2), 34 (1985).
One disadvantage of post-acceleration detectors, related to the energy of the electrons impinging on the final detector surface, represents a form of "Catch-22" for detector efficiency. High dynode voltages are required to accelerate high mass ions to an energy sufficient to produce secondary electrons, however, for high dynode voltages, the secondary electrons impinge on the multiplier with energies higher than the energy for maximum detection efficiency. This is illustrated by FIG. 3 which shows the CEM response as a function of the incident electron energy. (Taken from E. Kurz, Channel Electron Multipliers, American Laboratory (March 1979).) Inspection of FIG. 3 shows that for electrons of energy E=40 KeV, the detection efficiency has dropped to approximately 60% from a peak of 90% at E=500 eV. Therefore, the gain in secondary emission at the conversion dynode is offset in part by the decrease in detector efficiency at the higher incident electron energies.
Another disadvantage of post acceleration, ad other detectors, is that to detect negative sample ions, existing detectors must rely on the ejection of lower yield, secondary positive ions. Consequently the detection of high mass, negative ions is usually less sensitive than the detection of positively charged high mass ions. One approach to a post acceleration positive ion detector is shown in U.S. Pat. No. 4,423,324 to Stafford.
Various other approaches have been attempted to resolve one of more of these problems. E.g. N. R. Daly, Scintillation Type Mass Spectrometer Ion Detector, Rev. Sci. Instrum., 31, 264 (1960); I. Katakuse, H. Nakabushi, T. Ichhhara, Y. Fujita, T. Matsuo, T. Sukurai and H. Matsuda, Post Acceleration For Heavy Molecule Ion Detector, Mass Spectrometry, 33, 145 (April, 1985). The usefulness of such approaches for yielding effective high mass resolution has not been demonstrated, however.
Thus, prior art efforts to increase the efficiency of high mass ion detection in mass spectrometry instruments have been directed to post dynode signal amplification techniques and to increasing ion acceleration voltages at the source.